【打印本页】 【下载PDF全文】 【HTML】 查看/发表评论下载PDF阅读器关闭

←前一篇|后一篇→

过刊浏览    高级检索

本文已被:浏览 4073次   下载 2460 本文二维码信息
码上扫一扫!
带核定位信号的RARα与谷氨酸氨连接酶蛋白相互作用的胞内外验证
吴燕,刘北忠*,王翀,钟梁,朱丹,王春光,金丹婷
0
(重庆医科大学医学检验系临床检验诊断学教育部重点实验室,重庆 400016)
摘要:
目的 通过胞内外实验验证谷氨酸氨连接酶(GLUL)与带有核定位信号的维甲酸受体α(NLS-RARα)之间的相互作用。方法 将表达GLUL靶蛋白和NLS-RARα诱饵蛋白的两种重组表达质粒共同转化入AH109酵母菌,采用一对一的酵母双杂交技术验证它们在活细胞内的相互作用;通过构建GLUL及NLS-RARα蛋白标签融合表达载体,共转染至人胚肾HEK 293细胞,利用免疫共沉淀技术在细胞外验证它们之间的相互作用。 结果 GLUL靶蛋白和NLS-RARα诱饵蛋白质粒共转化AH109酵母菌后,可见蓝色的阳性克隆;GLUL蛋白及NLS-RARα标签融合表达载体构建成功,共同转染至HEK 293细胞,采用抗HA多克隆抗体免疫沉淀HA-NLS-RARα相互作用蛋白复合物后,抗c-Myc单克隆抗体免疫印迹检测,检测到GLUL-cMyc蛋白。结论 采用酵母双杂交和免疫共沉淀技术成功地在胞内外验证了GLUL与NLS-RARα之间存在特异性的相互作用。
关键词:  维甲酸受体α  核定位信号; 谷氨酸氨连接酶  蛋白质相互作用  双杂交系统技术  免疫共沉淀
DOI:10.3724/SP.J.1008.2010.0468
投稿时间:2009-11-30修订日期:2010-04-19
基金项目: 国家自然科学基金(30300449),国家中医药管理局面上项目(02-03ZP52),重庆医科大学课题(XBYB2007104).
Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells
WU Yan, LIU Bei-zhong*, WANG Chong, ZHONG Liang, ZHU Dan, WANG Chun-guang, JIN Dan-ting
(Key Laboratory of Clinical Medical Diagnostics, Ministry of Education, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China)
Abstract:
Objective To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.
Key words:  retinoic acid receptor α  nuclear localization signals  glutamate-ammonia ligase  protein interaction  two-hybrid system techniques  co-immunoprecipitation